Janus kinases (JAKs), members of the non-receptor protein tyrosine kinase family, are key components of signaling pathways in hematopoiesis and cellular immunity. JAKs are associated with the cytoplasmic domains of cytokine receptors and, upon cytokine-mediated receptor dimerization or rearrangement of pre-existing receptor dimers, are activated through trans-phosphorylation. Activated JAKs phosphorylate STATs (signal transducers and activators of transcription), which translocate to the nucleus and serve as transcriptional activators. Mammalian JAKs (JAK1-3 and TYK2) possess four domains in common: an N-terminal FERM domain, an SH2-liske domain, a pseudokinase domain, and a C-terminal tyrosine kinase domain. Extensive biochemical data, as well as gain-of-function mutations that cause myeloproliferative neoplasms (MPNs) in humans, have implicated the pseudokinase domain of JAKs as crucial for maintaining a low basal level of tyrosine kinase activity. There is also evidence that the pseudokinase domain plays a positive regulatory role in normal JAK2 activation (by cytokine) and in the hyperactivity of mutants such as V617F, the most prevalent MPN mutant. The first goal of this proposal is to employ structural, biochemical, and computational approaches to elucidate the molecular mechanisms that govern JAK2 activation, both normally (through cytokine) and pathogenically (through mutation), and receptor interaction. A second goal is to use this structural knowledge to discover novel allosteric inhibitors that could selectively inhibit JAK2 V617F. Such compounds would have potential use as therapeutics in the treatment of MPN patients.